Arginine-Modified 3D-Printed Chromatographic Supports

The increasing progression of biopharmaceutical-based therapies highlights the demand for efficient chromatographic methods that can be used to purify the desired biomolecules (e.g., nucleic acids, enzymes, or monoclonal antibodies) which are presently under consideration in clinical trials or approved by the Food and Drug Administration. These molecules present distinct chemical and structural properties, which are critical cues for the development and production of adequate chromatographic supports. Until now, it has not been possible to fully control the characteristics of the chromatographic matrices to assure the total reproducibility of their structure and packing. Meanwhile, three-dimensional printing (3DP) is in the early stage of its use in the production of chromatographic supports as a fast, very precise, and reproducible methodology. Although 3DP can provide excellent performance properties to the chromatographic structures, it cannot, per se, lead to high-quality pharmaceutical products. However, the association of affinity ligands, such as amino acids, which is possible in 3DP, could enable the attainment of high-purity yields of the desired molecules. Beyond the amino acids most widely studied as chromatographic ligands, arginine has been successfully immobilized on different chromatographic supports (namely, agarose bead matrices, macroporous matrices, and monoliths) to achieve extra-pure gene therapy products. In this research, we studied the immobilization of arginine on 3DP chromatographic supports, evaluating the stability of the ligand/chromatographic support linkage under different chromatographic conditions to determine the robustness of these new prototypes. Moreover, we also applied plasmid DNA samples to these supports to observe the practical behaviour of the developed arginine 3DP chromatographic structures.

Introduction
Chromatography is a widely used technique employed to separate and assess the chemical purity of drug substances, metabolites, and biological products in the pharmaceutical, food, and environmental fields, possessing great importance in quality control laboratories. The available chromatographic supports are based on microparticulate or monolithic structures, in which it is difficult to accomplish an ordered packaging. This may lead to deleterious effects on the chromatographic run efficiency. In general, this inability to strictly control the morphology and porosity of traditionally manufactured materials can result in low levels of reproducibility, with the frequent need to prepare and validate each column individually [1]. Additionally, since the performance of chromatographic separations depends on several factors, including the flow of the mobile phase within the column, with its influences on the axial dispersion and peak widening (resulting in low degrees of purity), scientific studies have shown that the use of ordered media provides a significantly improved chromatographic performance [2]. To solve this problem, additive manufacturing (AM) can be applied as a fast, highly accurate, and reproducible methodology for producing 3DP chromatographic structures. AM enables the effective control of the size, shape, position, alignment, and configuration of the structure to create complex structures that are impossible to produce conventionally [3]. The use of AM is expected to improve the performance of the separation, resolution, productivity, and cost-efficiency of chromatographic supports.
Included among a wide range of AM techniques is fused deposition modelling (FDM), which is a process based on the extrusion of a thermopolymer through a printing nozzle to produce a layer-by-layer construction. This printing methodology has already been successfully applied by other researchers for the printing of chromatographic structures. For example, Abdulhussain et al. (2020) used different polypropylenes to print chromatographic structures through FDM, which were used for the separation of different small molecules [4].
Although this potential to provide the structural organization and, consequently, reproducibility of the chromatographic supports, it is well known that the coupling of specific ligands with these structures is crucial for achieving extra-pure biological products, which is a basic requirement needed to fulfil the regulatory agencies' criteria [5]. Based on this, several affinity ligands, namely amino acids, have been successfully applied to the purification of biomolecules by chromatography. This kind of ligand can mimic the natural interaction phenomena that occur between nucleic acids and proteins in biological environments. Until now, amino acids such as arginine, histidine, and methionine, among others, have been successfully applied for the isolation of the biologically active supercoiled (sc) pDNA isoform [6].
In this work, we applied a new approach to produce reproducible and architecturally controlled chromatographic supports (which is not possible in the case of conventionally produced chromatographic supports). This new approach is based on additive manufacturing (AM) technology-3D printing. Additionally, arginine was immobilized in these 3D-printed structures that were then evaluated to assess the effective linkage between the amino acid and the chromatographic structure by SEM, FTIR, and EDX. Moreover, additional experiments were performed to determine whether the arginine attached to the scaffolds would disconnect when it came into contact with different buffers to assess the stability and robustness of the structures. Finally, pDNA adsorption experiments were conducted to explore the capacity of these novel chromatographic matrices to interact with the given biomolecules.

Production of the Chromatographic Scaffolds
The chromatographic scaffolds used in this work were produced using a BioExtruder device, developed at the Polytechnic of Leiria (P.L., Pt). This equipment enables the layer-by-layer extrusion of the molten PCL through a nozzle (23 gauge), with a 0/90 • laydown pattern previously designed by a computer. Therefore, cylindric chromatographic structures with a 10 mm diameter, 2.6 mm height, and an interconnected porous network of square channels were obtained.

Immobilization of Arginine in the Chromatographic Scaffolds
The arginine immobilization in the 3D-printed chromatographic structures was performed using three distinct steps. First, each scaffold was placed in a small glass vial, to which we added 3M NaOH, and then the vials were incubated at 37 • C, 100 rpm, for 5 h. Then, the NaOH was removed and the scaffolds were washed with Milli-Q H 2 O before the addition of 17 µL of epichlorohydrin, followed by incubation at 37 • C, 100 rpm overnight. In the third and final step, the epichlorohydrin was removed through washing with Milli-Q H 2 O, and then an arginine-saturated solution prepared in 2 M NaHCO 3 was added to each vial and left incubating at 37 • C, 100 rpm, for approximately 72 h. Note that the scaffolds were completely covered by the solutions in the vials. Afterward, the arginine-saturated solution was washed with Milli-Q H 2 O, and the scaffolds were stored in 20% ethanol until further use. After the 72 h incubation period, samples of the initial arginine-saturated solution from each vial were obtained for OD 235 nm measurements using a spectrophotometer (BMG LABTECH, SPECTROstarNano, Offenburg, Germany) to evaluate the amount of arginine immobilized.

Characterization of the Arginine-Modified 3DP Chromatographic Structures
To confirm whether the arginine immobilization in the scaffolds had been successful, different techniques were used. Non-immobilized scaffolds were used as the blank in all the techniques, and an n = 4, at the least, was considered.

Fourier Transform Infrared Spectroscopy (FTIR)
To progress from the immobilization process and, ultimately, identify evidence of the successful immobilization of the arginine, an FTIR analysis was performed. Briefly, a piece of the scaffolds was mounted on a diamond window and compressed to improve the spectrum signal-to-noise ratio. For each sample, 128 scans were acquired within a spectral width ranging from 4000 to 500 cm −1 and a spectral resolution of 4 cm −1 [7]. The non-immobilized matrix was used as the blank. The spectra were recorded using an FTIR spectrophotometer (Bruker, Alpha P, Bremen, Germany).
Scanning Electron Microscopy (SEM) and Energy-Dispersive X-ray Analysis (EDX) SEM (Vega3 Tescan equipment from Tescan, Brno, Czech Republic)) was used to evaluate any possible morphological changes that occurred during the arginine immobilization process. For this test, samples of the scaffolds were analyzed and compared under 15 kV and at variable magnifications. The samples were attached to a brass stub with doublesided tape and coated with a gold/palladium (Au/Pd) thin film by sputtering, using the sputter coater equipment (Quorum Technologies).
Additionally, elemental analysis was performed on both the immobilized and nonimmobilized scaffolds to confirm whether nitrogen (N) was present, as this can indicate the presence of arginine in the samples. To achieve the variation in the N atoms resulting from the arginine immobilization process, the samples were placed on aluminium stub supports and air-dried at room temperature, and then an EDX analysis (Bruker, XFlash Detector 6|30 from Karlsruhe, Germany) was performed.

Micro-Computed Tomography Analysis (µ-CT)
The extruded scaffolds were scanned using a Skyscan 1174 (Brucker, Karlsruhe, Germany) with an image pixel size of 30.11 µm, exposure time of 7000 milliseconds, and a rotation step of 1 • . The three-dimensional reconstructions were obtained using CTan and CTvox, while the transversal plane views were obtained using a data viewer. Porosity measurements (n = 3) were derived from the µ-CT reconstructions.

Evaluation of Arginine Leakage under Different Conditions
After proving the success of the arginine immobilization process, some experiments were carried out to ensure that the arginine was not easily released and washed out from the scaffolds, a process usually known as ligand leakage. Batch experiments using different buffers were thus performed to assess the stability of the immobilization. The selection of the buffers relied on the conditions that are commonly used for arginine chromatographic supports in the purification of nucleic acids, including the conditions required for support regeneration (0.1 M NaOH, used in regeneration/sanitization protocols; 2 M NaCl and 10 mM Tris-HCl, pH 8, used in the chromatographic runs; 20% ethanol, used in the storage of the scaffolds; water, used to clean the chromatographic support). For the experiments, each scaffold was placed in a glass vial, to which 1 mL of the selected buffer was added. The vials were incubated at room temperature at 100 rpm for 30 min. After incubation, the samples were analyzed by measuring the OD 235nm to evaluate whether the arginine was desorbed from the scaffolds. A comparison with non-functionalized chromatographic supports in contact with the same buffers was also performed.

Plasmid DNA Production and Isolation
The amplification of pcDNA3-FLAG-p53 was achieved using cell cultures of E. coli DH5α. The bacteria cultures were developed at 37 • C, 250 rpm in Erlenmeyer flasks with 500 mL of Terrific Broth medium (20 g/L of tryptone, 24 g/L of yeast extract, 4 mL/L of glycerol, 0.017 M KH 2 PO 4 , 0.072 M K 2 HPO 4 ) supplemented with 30 µg/mL ampicillin. Then, E. coli cells were grown until the late log phase (OD 600nm ± 9) and then collected by centrifugation at 4000 rpm and stored at −20 • C until further use.
To recover the produced pDNA, the E. coli cells were first disrupted by alkaline lysis, and the p53-encoding pDNA (sc and oc isoforms) was pre-purified using the NZYTech kit according to the manufacturer's instructions. Briefly, after the alkaline lysis, the pDNA molecules were bound to the NZYTech anion exchange resin under the appropriate low-salt and pH conditions. Then, the impurities were removed by a medium salt wash and, finally, the pDNA was eluted through the increase in ionic strength.

The pDNA Batch Adsorption Experiments
For the experiments, performed in batch conditions, each scaffold was placed in a glass vial, to which we added 2 mL of ((40) µg/mL) pDNA, and then the vials were incubated at 20 • C at 100 rpm for 15 min. Then, the pDNA samples were collected, and the vials were quickly washed with 1 mL of 10 mM Tris-HCl (pH 8). After the washing step, 2 mL of 2 M NaCl (pH 8) was added to the vials to induce the elution of the retained pDNA in the scaffold, followed by incubation at 20 • C at 100 rpm for 15 min. After incubation, the samples were collected. To evaluate the yield of recovered pDNA, OD 260nm measurements of the collected samples (in the various steps) were performed and compared with the initial pDNA solution, discounting the blank 10 mM Tris-HCl and 2 M NaCl buffers. This assay was performed in four independent assays on 4 different days.

Agarose Gel Electrophoresis
After assessing the binding profile, agarose gel electrophoresis was performed to verify whether pDNA was bound to, and recovered from, the chromatographic scaffolds. Thus, a 0.7% agarose gel stained with GreenSafe dye (NZYTech, Lisbon, Portugal) was prepared in 1X TAE buffer. Before loading the samples onto the gel, the samples recovered in the elution step (with NaCl) were subjected to a concentration process (Sartorius, Gottingen, Germany) to enable better visualization of the gel. A DNA ladder (ladder III, NZYTech) and the samples (16 µL of sample + 4 µL of loading buffer (3 mL of 30% glycerol, 25 mg of bromophenol blue (0.25%, PanReac AppliChem, ITW Reagents, Chicago, IL, USA), and Milli-Q H 2 O until it reached 10 mL)) were loaded onto the gel. Finally, electrophoresis was run with 1X TAE buffer (20 mM acetic acid, 1 mM EDTA, and 40 mM Tris base, pH 8.0) at 100 V for around 40 min, and the resulting gel was observed under UV light in a transilluminator (SmartDoc™, Accuris Instruments, Edison, NJ, USA).

Results and Discussion
The AM method used to produce the chromatographic structures was FDM. This is an economic and easy-to-use 3DP methodology with the ability to create structures of various shapes, with a low-price instrument, no need for chemical post-processing, no resins to cure, and a broad range of thermoplastics that can be successfully applied to the 3DP pieces [4]. In this work, the polymer chosen for the printing of the chromatographic structure was PCL, a material that is very easy to handle, with a slightly low fusion temperature (around 60 • C) that enables the achievement of robust pieces [8]. This is an important parameter to consider since due to the flow rate or the type of sample used, the pressure achieved during the chromatographic run can reach several "psi", leading to chromatographic structure damage [9].

Chemical Evaluation of the Immobilization Process of Arginine
One of the critical aspects of chromatographic support production is the effective immobilization of the desirable ligands. In this process, one must assure the stable linkage of the ligands to the chromatographic structure and its modification with a large number of ligands to reach the highest ligand density possible [10]. These two features significantly contribute to the support's robustness and increased binding capacity, which is very important for the purification results.
In this work, the immobilization process included three key steps, with the first comprising the PCL scaffold's immersion in NaOH, leading to the formation of hydroxyl (OH) and carboxylic (COOH) groups on the PCL filament surface; the second step comprising the use of epichlorohydrin to enrich the structure with epoxy groups (binding to the previously added -OH groups); and the third and final step is responsible for the linkage of the arginine to the 3D-printed chromatographic structure ( Figure 1) [11].
in the elution step (with NaCl) were subjected to a concentration process (Sartorius, Gottingen, Germany) to enable better visualization of the gel. A DNA ladder (ladder III, NZYTech) and the samples (16 µL of sample + 4 µL of loading buffer (3 mL of 30% glycerol, 25 mg of bromophenol blue (0.25%, PanReac AppliChem, ITW Reagents, Chicago, IL, USA), and Milli-Q H2O until it reached 10 mL)) were loaded onto the gel. Finally, electrophoresis was run with 1X TAE buffer (20 mM acetic acid, 1 mM EDTA, and 40 mM Tris base, pH 8.0) at 100 V for around 40 min, and the resulting gel was observed under UV light in a transilluminator (SmartDoc™, Accuris Instruments, Edison, NJ, USA).

Results and Discussion
The AM method used to produce the chromatographic structures was FDM. This is an economic and easy-to-use 3DP methodology with the ability to create structures of various shapes, with a low-price instrument, no need for chemical post-processing, no resins to cure, and a broad range of thermoplastics that can be successfully applied to the 3DP pieces [4]. In this work, the polymer chosen for the printing of the chromatographic structure was PCL, a material that is very easy to handle, with a slightly low fusion temperature (around 60 °C) that enables the achievement of robust pieces [8]. This is an important parameter to consider since due to the flow rate or the type of sample used, the pressure achieved during the chromatographic run can reach several "psi", leading to chromatographic structure damage [9].

Chemical Evaluation of the Immobilization Process of Arginine
One of the critical aspects of chromatographic support production is the effective immobilization of the desirable ligands. In this process, one must assure the stable linkage of the ligands to the chromatographic structure and its modification with a large number of ligands to reach the highest ligand density possible [10]. These two features significantly contribute to the support's robustness and increased binding capacity, which is very important for the purification results.
In this work, the immobilization process included three key steps, with the first comprising the PCL scaffold's immersion in NaOH, leading to the formation of hydroxyl (OH) and carboxylic (COOH) groups on the PCL filament surface; the second step comprising the use of epichlorohydrin to enrich the structure with epoxy groups (binding to the previously added -OH groups); and the third and final step is responsible for the linkage of the arginine to the 3D-printed chromatographic structure ( Figure 1) [11]. The surface of the PCL-printed chromatographic structures was firstly soaped in NaOH to obtain anchorage zones for the epoxy ring. Then, the PCL surface was activated using epichlorohydrin through a nucleophilic substitution reaction. Finally, the Larginine ligand was attached to the surface of the activated PCL structure by its amine group through the ring-opening base reaction [13].
Having completed the immobilization procedure, it was necessary to verify that the arginine was efficiently connected to the chromatographic support and, to achieve this, The surface of the PCL-printed chromatographic structures was firstly soaped in NaOH to obtain anchorage zones for the epoxy ring. Then, the PCL surface was activated using epichlorohydrin through a nucleophilic substitution reaction. Finally, the L-arginine ligand was attached to the surface of the activated PCL structure by its amine group through the ring-opening base reaction [13].
Having completed the immobilization procedure, it was necessary to verify that the arginine was efficiently connected to the chromatographic support and, to achieve this, different techniques, including FTIR, EDX, and UV spectroscopy, were performed ( Figure 2).
In Figure 2I, it is possible to observe the FTIR spectrum of the PCL structure before and after it came into contact with 3 M of NaOH for 4 and 5 h. Representative peaks of the PCL were observed at 2939 cm −1 (asymmetric CH 2 stretching), 2865 cm −1 (symmetric CH 2 ), 1718 cm −1 (carbonyl C=O stretching), 1188 cm −1 (C-O-H stretching), and 1163 cm −1 (C-O-C stretching) [14]. The peaks I-A (1567 cm −1 ) and B (1431 cm −1 ), which are presented in the samples within 4 h and 5 h of incubation with NaOH, can be related to the ester bond cleavage, demonstrating the PCL modification. Moreover, the sample modified during Pharmaceutics 2022, 14, 2266 6 of 12 5 h presented a higher peak intensity than the 4 h modified samples due to the higher modification level. Additionally, Figure 2IC shows an increase in the intensity of the C-O-H (carboxyl and hydroxyl) peak (1188 cm −1 ) compared to the intensity of the C-O-C (ester) peak (1163 cm −1 ) [15]. This intensity increase was more significant in the 5 h NaOH-treated than the 4 h NaOH-treated chromatographic supports due to the higher modification, which represents a larger number of available anchoring sites for epoxy group bonding.
Pharmaceutics 2022, 14, x FOR PEER REVIEW 6 of 13 different techniques, including FTIR, EDX, and UV spectroscopy, were performed ( Figure  2). Regarding the second step of the immobilization process, which is represented by the results presented in Figure 2II, it is possible to observe that both studied factors, namely, the quantity of epichlorohydrin and the contact time, influenced the efficiency of the epoxy groups' coupling to the PCL structure. The results show the number of nitrogen atoms (from the arginine) on the PCL structures incubated with different amounts of epichlorohydrin for 6 and 16 h. The data suggest that 16 h of contact with epichlorohydrin was more efficient in ensuring arginine coupling to the structure, while 17 µL was the ideal amount of this reagent required to promote greater arginine immobilization. However, even higher amounts of epichlorohydrin or increased contact times would not be beneficial for the immobilization process due to the corrosive properties of the reagent, which could degrade the PCL surface. The last step of the immobilization process comprised the binding of the arginine to the epoxy-activated chromatographic structure.
In Figure 2III, it is possible to observe the results obtained after the assessment of the N atoms of the arginine ligand in the internal region of the PCL chromatographic structure. It was observed that arginine linkage occurred in all the structures. However, on the surface, the amount of N atoms and, consequently, the amount of arginine were higher in comparison to the amount of these atoms present in the interior of the structure. This could be due to the porous dimension, which can lead to difficulty regarding the arginine ligands' ability to access the internal structure. To overcome this limitation, different immobilization conditions were used, specifically by increasing the contact time with the different immobilization solutions and increasing the mixing velocity during the process.
Comparing these results with others, the percentage of N atoms seemed to be diminished in our 3D-printed matrices [12]. However, it is important to note that this previously cited study does not refer to the ratio between the N atom and residual atoms, such as carbon, for example. This is an important step that must be performed since one can measure more dense spots, leading to an up-measurement of the N atoms.

Morphological Evaluation of the 3D-Printed Structures SEM
An in-depth analysis of the 3DP PCL structures before and after the immobilization process was also performed through SEM ( Figure 3) and µ-CT (Figure 4). This characterization was performed to evaluate the morphology, structure, piece robustness, filament alignment, and pore assessment and to guarantee the maintenance of the integrity/stability of the printed structures after the immobilization process.
The SEM analysis ( Figure 3) showed that, although the immobilization process could aggressively impact the printed structure, no degradation was observed, independently of the ampliations used in this analysis. This may indicate that the experimental conditions used for the arginine immobilization are appropriate and do not damage the chromatographic support. This is extremely important since the degradation of the surface can be an issue affecting the lifetime of the chromatographic structures in general [16].
Through the analysis performed in this study, it was also possible to measure the diameter of the filaments, which was around 342.2 ± 14 µm, a slightly higher value compared with the diameter of the extrusion needle. Moreover, the dimensions of the pores were also assessed, reaching an average value of 274 ± 4 µm. This porosity enabled the buffers and samples to flow through the internal and external structure of the 3DP chromatographic structure. These dimensions can lead the 3D-printed structures to acquire some of the advantages of the macroporous conventional supports, such as the ability to improve the access of pDNA to the internal voids and to enable the convective pore flow, consequently improving the internal mass transfer [12,17].
When compared with other types of chromatographic structures, such as the microparticulated structures, the beads can range from 30 to 500 µm and the pore size can range from 30 µm to 20 nm. Concerning the monolithic supports, the fiber usually ranges from 0.7 to 2 µm and the pore size average is 4.5 µm. Among these parameters, one of the most important to consider when designing the chromatographic piece is the pore dimensions, since the average pore size needs to be at least the same size as the radius of gyration of a pDNA molecule [18].  The SEM analysis ( Figure 3) showed that, although the immobilization process could aggressively impact the printed structure, no degradation was observed, independently of the ampliations used in this analysis. This may indicate that the experimental conditions used for the arginine immobilization are appropriate and do not damage the chromatographic support. This is extremely important since the degradation of the surface can be an issue affecting the lifetime of the chromatographic structures in general [16].
Through the analysis performed in this study, it was also possible to measure the diameter of the filaments, which was around 342.2 ± 14 µm, a slightly higher value compared with the diameter of the extrusion needle. Moreover, the dimensions of the pores were also assessed, reaching an average value of 274 ± 4 µm. This porosity enabled  The SEM analysis ( Figure 3) showed that, although the immobilization process could aggressively impact the printed structure, no degradation was observed, independently of the ampliations used in this analysis. This may indicate that the experimental conditions used for the arginine immobilization are appropriate and do not damage the chromatographic support. This is extremely important since the degradation of the surface can be an issue affecting the lifetime of the chromatographic structures in general [16].
Through the analysis performed in this study, it was also possible to measure the diameter of the filaments, which was around 342.2 ± 14 µm, a slightly higher value compared with the diameter of the extrusion needle. Moreover, the dimensions of the pores were also assessed, reaching an average value of 274 ± 4 µm. This porosity enabled

µ-CT
To better assess the internal morphology of the 3DP structure and achieve the exact values of the porosity and percentage of the pores, µ-CT was used (Figure 4). This technique is extremely valuable because it helps researchers to achieve the parameters previously described without cutting or manipulating the studied samples.
Through the analysis of these projections, it is possible to observe that the 3D-printed support comprises interconnected channels that lead to an interconnected structure, which is a key point for this kind of 3D structure that must be applied in the chromatographic field.
Additionally, through the acquired projections, it was possible to determine the structure's porosity as around 87% ± 4.3 (n = 3).
Comparing these results with the conventionally used/produced chromatographic structures, it is possible to observe that the obtained values are higher than the range of the values produced up to now. Therefore, in a microparticulate chromatographic structure, the external porosity is typically 40%, with values ranging between 37% and 42%. The porosity of the macropore network, which, in monolithic columns, plays the same role as that which the external or interparticle pore network plays in packed columns, is typically around 70% [18].

Assessment of the Immobilization Process Quality
Guaranteeing the effective linkage of the ligand to the chromatographic support is extremely important for the chromatographic process, as it determines the efficiency of the target biomolecules' retention, also influencing the maximum binding capacity of the support, with an impact on the recovery yield. In this regard, when we have a solid 3D structure, it is important to assure that the immobilization process is efficient not only on the surface of the piece but also on the internal part of the structure, which, due to the geometry of the 3D-printed structure, is usually more difficult to access. In this regard, in Figure 5, it is possible to observe the loss of arginine after the 3D-printed structures came into contact with different buffers. previously described without cutting or manipulating the studied samples.
Through the analysis of these projections, it is possible to observe that the 3D-printed support comprises interconnected channels that lead to an interconnected structure, which is a key point for this kind of 3D structure that must be applied in the chromatographic field. Additionally, through the acquired projections, it was possible to determine the structure's porosity as around 87% ± 4.3 (n = 3).
Comparing these results with the conventionally used/produced chromatographic structures, it is possible to observe that the obtained values are higher than the range of the values produced up to now. Therefore, in a microparticulate chromatographic structure, the external porosity is typically 40%, with values ranging between 37% and 42%. The porosity of the macropore network, which, in monolithic columns, plays the same role as that which the external or interparticle pore network plays in packed columns, is typically around 70% [18].

Assessment of the Immobilization Process Quality
Guaranteeing the effective linkage of the ligand to the chromatographic support is extremely important for the chromatographic process, as it determines the efficiency of the target biomolecules' retention, also influencing the maximum binding capacity of the support, with an impact on the recovery yield. In this regard, when we have a solid 3D structure, it is important to assure that the immobilization process is efficient not only on the surface of the piece but also on the internal part of the structure, which, due to the geometry of the 3D-printed structure, is usually more difficult to access. In this regard, in Figure 5, it is possible to observe the loss of arginine after the 3D-printed structures came into contact with different buffers. In Figure 5, it is possible to observe that the first washing step led to a loss of 20 mg of the ligand, which could be due to the washing of free arginine that was not fully linked to the PCL structure. It is also possible to observe that, in the following washing steps, only residual losses of this ligand could be verified, being extremely favourable for the chromatographic process since, at least in theory, it can promote reproducible results over the chromatographic support's lifetime. Therefore, we can conclude that nearly 70% of the initially calculated amount of arginine immobilized was still immobilized on the chromatographic piece.
Another important piece of information that can be observed from the analysis of Figure 5 is related to the use of different buffers in this 3DP chromatographic matrix immobilized with arginine ligands. From these results, it is possible to observe that the loss of ligands is residual/null in the majority of the cases. The only buffer that leads to a slight increase in the arginine loss is NaOH, when it is applied. NaOH is usually applied to promote a deeper cleaning of the chromatographic structures, mainly to guarantee their reuse [19]. However, using NaOH can damage the ligand over repeated cycling, leading to a decrease in the dynamic binding capacity, which is why the chromatographic resin suppliers recommend using this buffer only when extensive regeneration is needed [20,21]. In any case, concerning the initial amount of amino acid retained on the 3DP PCL structure, each cycle of NaOH leads to an arginine loss of 1.1 %, which, in theory, means that 24 NaOH cycles are needed for a 50% loss in the arginine ligands' linkage to the PCL support.

The pDNA Adsorption Ability
Arginine was selected for immobilization in the 3D-printed chromatographic supports mainly because it has already been successfully applied in the past for the selective recovery of different biomolecules and types of supports [12,[22][23][24]. Therefore, it is crucial to evaluate the chromatographic support's ability to interact with the target sample during the support development process.
Concerning this, the developed chromatographic support was placed in contact with a pDNA pre-purified sample (sc + oc isoforms), and the ability to interact with this biomolecule was evaluated. Since the arginine ligand has already been described as an affinity ligand that can promote multiple non-covalent interactions, it is possible to modulate the performed interactions with the target sample by manipulating the chromatographic conditions, namely the salt used. In this work, we decided to use NaCl to exploit mainly ionic interactions (since this kind of interaction between arginine and pDNA samples has provided good results [12,25]). The agarose analysis of this experiment is presented in Figure 6 where is possible to observe the profile of each peack of the purification assay. Additionally, from a complementary analysis was also possible to measurea total of 0.6 µg/mm 3 pDNA adsorbed on the 3D-printed chromatographic support. Therefore, it is worth mentioning that the arginine ligand enables the establishment of a multitude of interactions with the sc pDNA, such as hydrophobic, electrostatic, and cation-π interactions, van der Waals forces, and/or hydrogen bonds. Van der Waals forces and water-mediated bonds can contribute to the amino acid-DNA interactions and studies have shown that the arginine specificity is mainly due to hydrogen bonds, which are favoured by bidentate and complex interactions, preferentially with the guanine base [26].
Considering the obtained results, it is suggested that a larger chromatographic piece is needed for an increased level of pDNA binding. However, it is also relevant to mention that the great porosity and the contribution of the macropores can provide these 3D- Figure 6. Analysis of the agarose gel electrophoresis of samples collected from each peak in the purification assay. Lane M: molecular weight marker; Lane 1: feed sample (E. coli lysate pre-purified with a commercial kit); Lane 2: pDNA + Tris 10 mM pH 8 chromatographic support; Lane 3: pDNA + NaCl 2M pH 8 chromatographic support. Therefore, it is worth mentioning that the arginine ligand enables the establishment of a multitude of interactions with the sc pDNA, such as hydrophobic, electrostatic, and cation-π interactions, van der Waals forces, and/or hydrogen bonds. Van der Waals forces and water-mediated bonds can contribute to the amino acid-DNA interactions and studies have shown that the arginine specificity is mainly due to hydrogen bonds, which are favoured by bidentate and complex interactions, preferentially with the guanine base [26].
Considering the obtained results, it is suggested that a larger chromatographic piece is needed for an increased level of pDNA binding. However, it is also relevant to mention that the great porosity and the contribution of the macropores can provide these 3D-printed chromatographic structures with higher permeability and higher efficiency than those of the conventional columns. Moreover, it is possible to use more dense extracts without compromising the flow within the 3D-printed chromatographic structure and, additionally, it is possible to operate these matrices at higher velocities to carry out faster analyses.

Conclusions
As mentioned before, chromatographic approaches must be improved in such a way that the chromatographic bed can be fully organized to guarantee its reproducibility and enable the prediction and the full computational simulation of the chromatographic process. To achieve this, and to create exceptional chromatographic models that can overcome the existent models on the market, a mutual effort between scientists and industry is needed.
Therefore, 3D printing has a panoply of applications, and its use to produce chromatographic structures remains little explored. In light of this, in this research work, we presented the production and development of a 3D-printed chromatographic structure designed to be applied in affinity chromatography.
We demonstrated the successful production of the PCL matrix using FDM, and then arginine ligands were successfully immobilized on its surface. Several experiments were performed to guarantee the stability of the linkage between the chromatographic support and arginine using complementary techniques and different buffers (the buffers were chosen taking into account those that are commonly applied in chromatographic experiments and the sanitation of a conventional arginine chromatographic support). The interaction between the ligand and the pDNA was also evaluated, and the 3D-printed chromatographic support was able to interact with the target sample. Overall, it was possible to produce arginine chromatographic supports that can be applied as pDNA adsorbers, with a fully controlled geometry, using 3D printing methodologies.
Concerning the developed structures, it is also interesting to note that the large pores enable the application of more dense extracts, without flogging or clogging, which creates the possibility of using these structures to recover different biomolecules/drugs. Moreover, with the achieved architecture and its easy manipulation/production ability, it may be possible to use these new supports for substance/biomolecule quantification. Additionally, since arginine has been used as a specific ligand, it is possible to guarantee that the 3Dprinted structure will enable the separation of other kinds of biomolecules [24]. Finally, since the developed 3D-printed support was successfully activated via epoxy, it can enable the immobilization of other ligands, exponentially increasing the range of application of the developed structure.

Conflicts of Interest:
The authors declare no conflict of interest.


Effect of a Diet Supplemented with Sphingomyelin and Probiotics on Colon Cancer Development in Mice

Previous studies have reported that dietary sphingomyelin could inhibit early stages of colon cancer. Lactic acid–producing bacteria have also been associated with an amelioration of cancer symptoms. However, little is known about the potential beneficial effects of the combined administration of both sphingomyelin and lactic acid–producing bacteria. This article analyzes the effect of a diet supplemented with a combination of the probiotics Lacticaseibacillus casei and Bifidobacterium bifidum (108 CFU/ml) and sphingomyelin (0.05%) on mice with 1,2-dimethylhydrazine (DMH)-induced colon cancer. Thirty-six BALB/c mice were divided into 3 groups: one healthy group (group C) and two groups with DMH-induced cancer, one fed a standard diet (group D) and the other fed a diet supplemented with sphingomyelin and probiotics (DS). The number of aberrant crypt foci, marker of colon cancer development, was lower in the DS. The dietary supplementation with the synbiotic reversed the cancer-induced impairment of galactose uptake in enterocyte brush–border–membrane vesicles. These results confirm the beneficial effects of the synbiotic on the intestinal physiology of colon cancer mice and contribute to the understanding of the possible mechanisms involved.

Introduction
Colorectal cancer (CRC) is the third most common diagnosed cancer representing an important health issue worldwide. Despite strong hereditary components, about 80% of cases of colorectal cancers are sporadic and develop slowly over more than 10 years. Epidemiologic studies have shown that the environmental factors, especially diet, are important risk factors for CRC [1,2] Mounting evidence supports the view that the colonic microbiota is involved in the etiology of colon cancer. Different factors, such as probiotics and different dietary bioactive compounds can modulate gut microbiota and its metabolism [3]. In this context, several investigations have focused on the beneficial effects of probiotics and bioactive compounds and their possible role in the prevention of colon cancer [4,5]. Most probiotics are members of the genus Bifidobacterium and several genera of the Lactobacillus group, but Saccharomyces and Enterococcus have also been studied. Experimental animal and human studies have shown that probiotics may reduce intestinal permeability and participate in the regulation of several intestinal functions [6][7][8]. Concretely, animal studies have demonstrated that certain species of lactic acid-producing bacteria, such as Lacticaseibacillus casei [9] and Bifidobacterium bifidum [10], could prevent colon cancer and other diseases linked to the gastrointestinal tract.
On the other hand, sphingolipids are structural and functional bioactive lipids found in eggs, milk, meat, fish, and soybeans that can act as chemo-protective agents regulating cell growth, differentiation, and death [11,12]. Sphingomyelin (SM) is the most abundant sphingolipid in plasma lipoproteins and contains predominantly a phosphocholine as head group [13]. The hydrolysis of SM by alkaline sphingomyelinase (alk-SMase) generates other bioactive molecules, such as ceramide and sphingosine, that play key roles in the maintenance of intestinal mucosal integrity and the inhibition of colon tumorigenesis [12,14]. Ceramide has been proposed as an important intracellular messenger in different signalling pathways implicated in the regulation of cellular proliferation, differentiation, and apoptosis [14][15][16]. Early studies have indicated that a therapy based on probiotics mixture containing several strains of acid lactic bacteria increased alk-SMase levels in mice with inflammatory gut disease [17] and cancer [18]. Furthermore, the combination of probiotic and other dietary compounds may exert additive effects in the improvement of colon carcinogenesis as compared to its separate administration [2,19]. However, the combination of SM and probiotics has not yet been analyzed although it could be hypothesized that dietary supplementation with probiotics together with SM could help to maintain the membrane integrity of the enterocytes, as well as alk-SMase activity in colon cancer models. Therefore, in this study, we have analyzed, for the first time, the beneficial effects of the combined administration of both lactic acid-producing bacteria (L. casei and B. bifidum) and SM in a mouse model of 1,2-dimethylhydrazine (DMH)induced colon cancer.

Bacterial Strains and Growth Condition
The L. casei CECT 475 T strain was isolated from kefir manufactured in the Lactology Laboratory of the Universidad Pública de Navarra, whereas the B. bifidum strain was obtained from the Spanish Collection of Cultures (CECT) with the number CECT 870. Both strains were used in this study as probiotics. Both bacteria were grown in autoclaved skim milk (Difco™; BD, Detroit, MI, USA), at 37 °C for 24 h under aerobic conditions for L. casei and at 39 °C for 72 h under microaerophilic conditions for B. bifidum in the presence of 5% CO 2 . The cell pellets were resuspended in 10% Difco™ skim milk at a final concentration of 10 8 CFU/ ml for each strain.

Chemicals
All chemicals were purchased from Sigma Chemicals (St. Louis, MO, USA) unless otherwise noted. All reagents were of analytical grade.

Animals, Diets, and Experimental Design
Thirty-six male BALB/c mice of 28 days old and weighing about 20 g were obtained from the colony of Charles River Laboratory Animals (Barcelona, Spain). The mice were housed in cages (four animals per cage) and kept in a wellventilated, thermostatically controlled room (22 ± 2 °C temperature and 55 ± 5% relative humidity) with a photoperiod of 12 h light/night cycle.
The animals, after an acclimatization period of 4 days, were weighed and randomly assigned to 3 homogeneous groups (n = 12, each) and fed a standard diet (AIN-93G, Research Diets Inc., New Brunswick, NJ, USA). Control group (C) animals were injected subcutaneously with ethylenediaminetetraacetic acid (EDTA) 1 mM, whereas the mice of the DMH group (D) and DMH + supplemented diet group (DS) were injected subcutaneously with 1,2-dimethylhydrazine (DMH) dissolved in EDTA 1 mM (30 mg DMH/ kg body weight, twice per week for 3 weeks) to induce the pre-neoplastic lesions (ACF).
Fresh diets were prepared weekly with purified ingredients. All diets were isonitrogenous and isoenergetic and were stored at 4 °C until serving feed. One week after the last DMH injection, the animals in the DS group were fed the standard diet supplemented with sphingomyelin 0.05% and skim milk was supplemented with probiotic bacteria at 10 8 CFU/ml. Food and drink were available ad libitum. The body weight was recorded weekly.
At the end of the 66th day of the experimental period, the animals were anesthetized with CO 2 and sacrificed by decapitation. Trunk blood was collected for the measurement of serum biochemical parameters, and different organs (spleen, liver, colon, jejunum, and cecum) were extracted and weighed. The jejunum was carefully removed, flushed out with ice-cold saline, frozen in liquid nitrogen and stored at −80 °C until processed for the isolation of the brush-border-membrane vesicles (BBMV) for the measurement of the intestinal absorption of D-galactose.
The Animal Research Ethics Committee of the "Universidad Pública de Navarra" reviewed and approved (reference PI:07/06) the animal care protocol and the killing method to ensure compliances with the guidelines of the Canadian Council on Animal Care [20].

Histological Analyses
Colon sections of 6 animals per group were flushed with icecold saline, opened longitudinally, fixed flat in 10% formalin for 24 h, dehydrated, and stained in 0.1% methylene blue for 15 min. The colon pieces were placed mucosa-side up on glass slides and evaluated for the presence of aberrant crypts foci by light microscopy (40 × or 100 ×). The aberrant foci were identified following the counting criteria described by Paulsen et al. [21]. Before being frozen, a portion of the distal colon (1 cm length) was taken for histological examination. Samples of distal colon tissue were immediately fixed in 4% formalin solution for 24 h. They were then dehydrated, embedded in paraffin, sliced into 5-µm sections and processed for hematoxylin-eosin staining.

Serum Biochemical Assays
All serologic parameters ( Table 2) were quantified in an automatic chemistry analyzer Cobas-Mira (Roche Diagnostic System, Basel, Switzerland) following the manufacturer's procedures.

Preparation of Mouse Intestinal BBMV
BBMV were obtained according to the method described by Shirazi-Beechey et al. [22]. Briefly, the BBMV were prepared from a portion of small intestine extracted from each animal of the three different experimental groups.
The mucosa was then resuspended in buffer containing 100 mM mannitol and 2 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES) adjusted to pH 7.1 with 1 M Tris-HCl buffer. The suspension was homogenized with a Potter-Elvehjem (Braun, Melsungen, Germany) at 3000 rpm at 4 °C for 1 min. Next, MgCl 2 was then added to the homogenate to a final concentration of 10 mM, and the mixture was maintained on ice with continuous ice-cold shaking for 20 min. After that, the mixture was centrifuged at 2000 g for 15 min, and the supernatant was collected and centrifuged at 27,000 g for 30 min. This supernatant was discarded and the pellet resuspended in a buffer containing 100 mM mannitol, 0·1 mM MgSO 4 and 2 mM HEPES adjusted to pH 7·4 with Tris. After a second precipitation with MgCl 2 , the mixture was finally centrifuged at 27,000 g for 30 min. The pellet was then resuspended in a buffer containing 300 mM mannitol, 0·1 mM MgSO 4 and 10 mM HEPES adjusted to pH 7·4 with Tris. The BBMV of ten mice were pooled, assayed for protein content by using the Bradford diagnostic kit (Bio-Rad Laboratories, Barcelona, Spain), diluted to 10 mg BBMV protein/ml, aliquoted and frozen in liquid nitrogen. The final BBMV preparation was fivefold enriched for sucrase specific activity compared with the initial homogenate.

Sugar Uptake by BBMV
Sugar uptake by the BBMV was measured using a slightly modified version of the rapid filtration technique developed by Hopfer [23]. Three replications were performed for each experimental group. D-galactose (0.1 mM) uptake was determined in the presence of a Na + gradient at pH 7.4 and 37 °C. BBMV were incubated in a medium containing 0.1 mM D-galactose, 100 mM NaSCN, 100 mM mannitol, 0.1 mM MgSO 4 , 10 mM HEPES, and traces of D-[1-14 C] galactose (0.037 MBq/ml; Amersham Radiochemical Centre, UK). At the different incubation times, uptake was halted by adding ice-cold stop solution (150 mM KSCN, 0.25 mM phloridzin and 10 mM HEPES) at pH 7.4 for the galactose uptake determination.
The suspension was poured immediately onto a cellulose nitrate filter (0.45 μm, 25 mm diameter; Sartorius, Edgewood, NY, USA) and the filter was then washed twice in ice-cold stop solution and dissolved in HiSafe 3 scintillation liquid for the final measurement of radioactivity using a β-counter (1450 MicroBeta® TriLux; Wallac, Turku, Finland).

Statistical Analysis
Descriptive and inferential statistics were used according to procedures described by Anderson [24]. The normality of the sample distribution of each continuous variable was tested with the Kolmogorov-Smirnov test. To identify significant differences among the groups (body and organ weights, and biochemical parameters), statistical analysis was performed by one-way ANOVA followed by Dunnett's test (D group as reference). Kruskal-Wallis followed by Mann-Whitney U test (D group as reference) were used to compare Western blot data and the ACF number of the three experimental groups. Differences were considered statistically significant when 2-tailed p < 0.05. Statistical calculations were performed with the statistical software package SPSS version 21.0 for Windows (SPSS, Chicago, IL, USA).

Animal Growth: Body and Organ Weights
The second week after cancer induction, body weight was significantly lower in the animals of the D and DS groups when compared with the C group (Fig. 1). The body weight of the animals of the DS group was significantly higher than those of the D group from day 45 of the experimental period, indicating that the dietary supplementation improves the growth rate of the animals.
Regarding the weights of the different tissues (Table 1), colon, and liver were the only organs that showed statistical differences when comparing the three experimental groups. Colon weight was higher in the DS group when compared with C (p = 0.002) and D (p = 0.015) groups, whereas liver weight was lower in D (p = 0.001) and DS (p = 0.007) groups when compared with C group.

Serum Biochemical Measurements
Serum glucose, urea, total cholesterol and high-density lipoprotein cholesterol levels tended to be higher in the D and DS groups, but without statistical differences ( Table 2). Serum aspartate aminotransferase levels were significantly lower in the control group and in the DS group when compared to D group although statistically significant differences were only observed in control group. In this line, alanine aminotransferase levels were also significantly lower in control and DS groups when compared to D group indicating that the induction of pre-neoplastic lesions in the colon increased the enzymatic activity and that the administration of the symbiotic combination reduced this increase, although control levels were not reached. This altered enzymatic activity observed in the DMH treated animals confirms the DMH-induced liver damage observed in treated animals. The supplementation with SM and probiotics did not prevent the liver weight loss observed in the DMH-treated mice, but it reverted the increase in alanine amino transferase serum level (p ≤ 0.05).

Histological Analysis
These ACF are considered indicators of pre-neoplastic damage. All the ACF found were located in the distal colon. Interestingly, dietary supplementation with sphingomyelin plus L. casei and B. bifidum resulted in a significantly reduction in the number of ACF when compared with the D group (Fig. 2). Colon sections of control mice displayed normal crypt foci and a colonic architecture with no signs of apparent abnormality. However, large areas with dense lymphocytic hyperplasia, compatible with preneoplastic lesions, were observed in the D group. Interestingly, this high lymphocyte infiltration was reduced by the administration of the symbiotic compound (Fig. 3).

Sugar Uptake in BBMV
Intestinal function has also been analyzed in the present work by measuring D-galactose uptake in BBMV. In this context, sugar uptake was stimulated by CRC development. The presence of the synbiotic in the diet inhibited D-galactose uptake as compared to DMH group (Fig. 4). This effect was observed only when the nutrients entered the enterocyte by an active transport mechanism that was mediated by a transporter located in the brush-border-membrane (short assay times and a Na + gradient). For longer incubation times (10 min and 60 min, respectively), the sugar entered the vesicles by a process of diffusion that was not altered by the synbiotic.

Discussion
Previous reports have demonstrated that several probiotics and bioactive compounds have a positive effect on the immune system, improving the immune response by unspecific and specific mechanisms [25]. In the context of colorectal cancer, the protective effect of dietary supplementation with different probiotics and synbiotics has been studied in animal models [26].
The present work has analyzed the effects of the combination of two probiotics (L. casei, B. bifidum) and a sphingolipid (sphingomyelin) on the physiopathology and intestinal function of mice with DMH-induced colon cancer. As there are no animal models that develop colon cancer spontaneously, DMH has been used to induce pre-cancerous lesions that are histologically similar to those observed in humans and has been widely used in the literature [27,28]. Regarding body weight, the results obtained are in agreement with other studies that did not find differences in body weight gain after supplementation with SM [11,15] or probiotics [6] and in line with studies by other authors in which DMH has been used as carcinogen [29]. Although sphingolipids have relevant biological activities, they are not considered as essential nutrients for an optimal growth rate of the animals [30,31].
As it has been previously mentioned, colon and liver were the only organs affected by the pre-cancerous lesion induction or synbiotic treatment. DMH-treated animals (D) showed a tendency to a higher colon weight than the untreated control group. This effect could be due to the initial stage of the disease that occurs with the presence of colon mucosa polyps that would increase the organ weight. However, the colon of the animals treated with the supplemented diet (DS group) was significantly higher than the one of the DMH group (D) suggesting that the consumed bacteria might attach to the colonic mucosae and increase, in consequence, the colon weight. In this sense, other authors have demonstrated that some strains of the Lactobacillus group are able to colonize the colon and adhere to the intestinal epithelium [32]. On the other hand, the liver was also affected by CRC development. Liver weight was lower in the animals with DMH-induced cancer than in the control group, suggesting that liver damage is related to colon cancer development and therefore could be implicated in the body weight loss observed in these animals. This liver damage was confirmed by the altered levels of serum alanine aminotransferase and aspartate aminotransferase observed in D and DS groups. In this context, a previous study found lower liver weight and decreased hepatic lipid accumulation in mice fed a diet supplemented with 0.2-0.4% sphingolipids [33]. Furthermore, other authors have shown that treatment with probiotics like Lactobacilli and Bifidobacteria could improve liver function [34].
Moreover, the dietary supplementation decreased the number of ACF as compared to D group, as found in  [37]. Finally, the inhibition of tumor formation due to dietary sphingomyelin has been attributed to a normalization of cell proliferation and rate of apoptosis, but not the induction of differentiation [12]. Finally, it seems that diet supplementation with the two bacteria and sphingomyelin inhibits the mechanisms involved in sugar absorption stimulation observed in DMH animals, restoring the sugar uptake levels obtained in control mice. These findings are in line with previous studies that demonstrated that probiotics [6] and sphingomyelin [11] are able to decrease glucose transport in intestinal epithelial cells models. These results suggest that the administration of the synbiotic could help to prevent colon cancer development in humans since the stimulation of sugar uptake increases the amount of intracellular glucose available for metabolic conversion, thereby promoting enhanced cell proliferation and cancer development [38].
In summary, the dietary supplementation with the synbiotic preparation reduced the number of histological intestinal lesions, suggesting that this combination has beneficial effects in DMH-treated mice with pre-neoplastic lesions. Moreover, this supplementation reversed the impaired intestinal sugar uptake observed in animals with DMH-induced pre-cancerous lesions, suggesting that it could be a good complementary therapy for the prevention of colon cancer in humans although more investigations are needed.


Single and Multiplex Immunohistochemistry to Detect Platelets and Neutrophils in Rat and Porcine Tissues

Platelet–neutrophil complexes (PNCs) occur during the inflammatory response to trauma and infections, and their interactions enable cell activation that can lead to tissue destruction. The ability to identify the accumulation and tissue localisation of PNCs is necessary to further understand their role in the organs associated with blast-induced shock wave trauma. Relevant experimental lung injury models often utilise pigs and rats, species for which immunohistochemistry protocols to detect platelets and neutrophils have yet to be established. Therefore, monoplex and multiplex immunohistochemistry protocols were established to evaluate the application of 22 commercially available antibodies to detect platelet (nine rat and five pig) and/or neutrophil (four rat and six pig) antigens identified as having potential selectivity for porcine or rat tissue, using lung and liver sections taken from models of polytrauma, including blast lung injury. Of the antibodies evaluated, one antibody was able to detect rat neutrophil elastase (on frozen and formalin-fixed paraffin embedded (FFPE) sections), and one antibody was successful in detecting rat CD61 (frozen sections only); whilst one antibody was able to detect porcine MPO (frozen and FFPE sections) and antibodies, targeting CD42b or CD49b antigens, were able to detect porcine platelets (frozen and FFPE and frozen, respectively). Staining procedures for platelet and neutrophil antigens were also successful in detecting the presence of PNCs in both rat and porcine tissue. We have, therefore, established protocols to allow for the detection of PNCs in lung and liver sections from porcine and rat models of trauma, which we anticipate should be of value to others interested in investigating these cell types in these species.

Introduction
Animal models of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) have provided useful insights into the contribution of platelets and neutrophils in the pathogenesis of these diseases, as such models are often investigated in rodents [1][2][3]. One important cause of ALI results from blast exposure, and there is a considerable body of evidence investigating this type of lung injury in large animals, such as pigs, as well as in rodents. Despite experiments being more costly and requiring scientists with highly specialized technical skills, porcine models of trauma allow for accurate laboratory recapitulation of trauma and blast injury, as their physiological similarities and size relative to humans enables direct testing of clinical protocols for the treatment of injury resulting from traumatic injury, including blast injury [4,5]. Blast injuries (resulting e.g., from explosions) are complex injuries, the components of which are characterised by the part of the explosion that caused them [4,5]. Primary blast injury is due to the shock wave, secondary blast injury is due to the fragments and debris energised by the explosion, and tertiary blast injury is due to the rapid movement of the casualty against other objects. Thus, porcine models of blast injury have enabled characterisation of the physiological changes that follow blast exposure, which includes bradycardia, hypotension and changes in Methods Protoc. 2022, 5, 71 2 of 18 respiratory parameters such as apnoea [6,7]. Furthermore, a complex injury model featuring blast injury (primary blast injury) and controlled haemorrhage (simulating the consequences of secondary blast injury), albeit without tertiary blast injury, has been developed and characterised and has already proven advantageous for informing military treatment protocols [8].
Due to lower costs and ease of handling and housing, rodent models have also been used to assess physiological outcomes following blast injury, and the availability of a wide range or reagents and tools has made rodents particularly beneficial for understanding different aspects of the inflammatory response associated with ALI, such as leukocyte recruitment [2,3,9,10], and following blast-and trauma-induced injuries [11][12][13][14].
Immunohistochemistry (IHC) is a very useful and widely used laboratory technique, allowing for the visualisation of in situ proteins in tissues following inflammatory insults [2,15]. However, most of the commercially available reagents used to carry out high quality IHC have been developed for murine, rather than rat experiments, and, of course, for investigating human tissues, including the lung [15]. There are, however, only a very limited number of reagents available for carrying out IHC in other species, such as the pig and rat. Given the usefulness of rats and pigs in investigating the physiological changes accompanying traumatic injury including blast injury, we have, therefore, evaluated a range of reagents that may be of value in these species for IHC of lungs and other tissues. In particular, given the involvement of platelets and neutrophils in lung injury [1][2][3]16], we have investigated a range of reagents for their ability to detect these important cell types immunohistochemically in the lungs and livers of rats and pigs.

Background to In Vivo Experimentation and Tissue Collection
All in vivo experiments reported in this study were carried out in the laboratories of DSTL, Porton Down, Salisbury, UK. All animal experiments were conducted following local ethical approval and in accordance with the Animals (Scientific Procedures) Act 1986 (United Kingdom). These studies were conducted under terminal anaesthesia (animals remained anaesthetised throughout the procedure and were killed humanely at the end of the study with an overdose of anaesthetic). Detailed procedural methodology is provided in previous reports for both the rat trauma and haemorrhagic shock protocol [17], as well as the severe injury and shock with blast exposure model in pigs [7,8,18].
Male rats (6-8 weeks old) were bred from an in-house colony of Porton Wistar rats and had access to chow (LabDiet EURodent Diet 22%) and water ad libitum. The pigs used in this study were female cross-bred large white pigs (43-56 kg), housed indoors, which were fed ad libitum on a complete diet comprising coarse ground mixture of wheat, barley, soya protein, vitamins, and minerals, with free access to water.

Tissue Collection
Rat protocol: six hours after the end of haemorrhage, as described elsewhere [17], all rats were killed humanely with an overdose of anaesthetic (Euthatal, Merial Animal Health Ltd., Harlow, UK). For FFPE lung samples, the accessory lobe was excised and immediately submerged in 4% formaldehyde for a minimum of 24 h. For frozen tissue, representative 1 cm 3 samples of the left and right lung lobes were flash-frozen in liquid nitrogen.
Pig protocol: pigs were anaesthetised, instrumented, injured, and resuscitated as previously described [18]. All animals received a soft tissue injury and 30% blood volume haemorrhage, but, in addition, a proportion of animals also received a whole-body blast exposure, as has been previously described [7]. Then, 4 h post injury, the pigs were humanely killed with an overdose of sodium pentobarbitone (Euthatal, Merial Animal Health Ltd., Bracknell, UK) given intravenously, and samples of lung and liver were collected post mortem for histological analysis and IHC. For FFPE lung tissue, a representative sample of the accessory lobe was excised and submerged in 4% formaldehyde for a minimum of 24 h, before undergoing standard histoprocessing into FFPE blocks. For frozen lung tissue, Methods Protoc. 2022, 5, 71 3 of 18 a representative 1 cm 3 piece was sampled from the left caudal lobe and was snap-frozen in liquid nitrogen.
Rat and pig lung tissues were subsequently provided to the Sackler Institute of Pulmonary Pharmacology at King's College London, where samples were sectioned and IHC protocol optimisation experiments were performed.
Frozen tissue samples were embedded into OCT and sectioned to 8 micrometres using a cryostat (frozen) or 4 micrometres using a microtome (FFPE) and stored at −80 • C or room temperature, respectively. The negative control was a consecutive tissue section that received all the immunohistochemistry reagents, except for the primary antibody, which enabled detection of unspecific reagent staining. On the day of IHC, slides were defrosted for 30 min. Sections were fixed for 10 min in 4% formaldehyde diluted in PBS. Endogenous peroxidases were blocked by using 3% hydrogen peroxide (H 2 O 2 ) solution. Tissue sections were blocked by pipetting 5% donkey serum in BSA onto tissue sections for 10 min. Donkey serum was removed before adding the primary antibody.

FPPE Tissue
For paraffin-embedded tissue, lung sections were removed of wax by submerging in 100% xylene for 10 min. Sections were rehydrated in decreasing concentrations of ethanol (100%, 90%, 70%) for 1 min in each. Endogenous peroxidases were blocked in the sections by using 3% hydrogen peroxide (H 2 O 2 ) solution. Sections then underwent heat-induced epitope retrieval (HIER) for 10 min at 100 degrees Celsius, using a microwave and a domestic pressure cooker. The HIER buffer was microwaved in a pressure cooker until it reached 100 • C, before the slides were added. The following HIER buffers were tested: 1. Tris-EDTA buffer [10 mM Trizma base (Sigma Aldrich, Poole, UK) 1 mM EDTA solution (Sigma Aldrich, Poole, UK), 0.05% Tween 20 (Sigma Aldrich, Poole, UK), pH 9.0]; 2. EDTA buffer [1 mM EDTA (Sigma Aldrich, Poole, UK), pH 8.0]; and 3. sodium citrate buffer [10 mM Sodium citrate (Fisher Scientific, Loughborough, UK), 0.05% Tween 20, pH 6.0]. Once the pressure dropped, the lid of the pressure cooker was removed, and slides were cooled under cold water for 5 min. Tissue sections were blocked by pipetting 5% donkey serum in BSA onto tissue sections for 10 min. Donkey serum was removed before adding the primary antibody.
Methodologies for IHC (frozen and FFPE): the appropriate primary antibody to detect rat neutrophils (Table 1), rat platelets (Table 2), porcine neutrophils (Table 3), or porcine platelets (Table 4), diluted in 5% donkey serum (in PBS), was added to lung sections and incubated at room temperature for 2 h. For multiplex IHC, primary antibodies were diluted together in 5% donkey serum in Tris-buffered saline (TBS), and the incubation step was for 1 h. Primary antibodies were washed off using PBS (or TBS for multiplex protocol). Excess PBS (or TBS) was removed, and the appropriate biotinylated secondary antibody was added (Table 5). Secondary antibody was incubated on sections for 1 h at room temperature. Secondary antibody was washed in PBS (or TBS) under gentle agitation for 5 min.

Rat Monoplex Horse Radish Peroxidase Detection and Colour Development
The Vectastain Elite ABC horseradish peroxidase (HRP) detection kit (PK-6100, Vector Laboratories, Burlingame, CA, USA) was prepared 30 min prior to use, in accordance with the instructions of the manufacturer (0.5% solution A and 0.5% solution B diluted in 5% donkey serum). Following washing, excess PBS was removed from slides, and the Vectastain Elite ABC HRP detection kit was pipetted onto each tissue section for 30 min. The Vectastain Elite ABC HRP reagent was then washed off in PBS under gentle agitation for 5 min. Sections were submerged in 3, 3 -diaminobenzidine (DAB) development buffer (0.1 M Trizma base (pH 7.6), 5% 3, 3 -DAB and 0.3% H 2 O 2 ) for 10 min. DAB forms a brown precipitate in regions of positive antigen detection.

Rat Multiplex Alkaline Phosphatase and Horse Radish Peroxidase Detection and Colour Development
ImmPRESS-AP Anti-Rabbit IgG (alkaline phosphatase) polymer detection kit (MP-5401, Vector Laboratories, Burlingame, CA, USA) was used at the recommended working concentration, in accordance with the instructions of the manufacturer. Following washing, excess TBS was removed and ImmPRESS-AP Anti-Rabbit IgG (alkaline phosphatase) polymer detection reagent was added to each tissue section. The AP reagent was incubated on tissue sections for 30 min at room temperature before being washed off using TBS under gentle agitation for 5 min.
The Vectastain Elite ABC horseradish peroxidase (HRP) detection kit (PK-6100, Vector Laboratories, Burlingame, CA, USA) was prepared 30 min prior to use, in accordance with the instructions of the manufacturer (0.5% solution A and 0.5% solution B diluted in 5% donkey serum). Following washing, excess TBS was removed and 100 µL of the Vectastain Elite ABC HRP detection kit was pipetted onto each tissue section for 30 min at room temperature. The Vectastain Elite ABC HRP reagent was washed off using TBS before being submerged in TBS under gentle agitation for 5 min.
For alkaline phosphatase dependent detection, the Vector Blue alkaline phosphatase (AP) substrate kit (SK-5300, Vector Laboratories, Burlingame, CA, USA) was used. This was prepared in accordance with the instructions of the manufacturer, with the addition of levamisole (SP-5000, Vector Laboratories, Burlingame, CA, USA), an endogenous AP inhibitor, that was added in accordance with the instructions of the manufacturer. Following washing, excess TBS was removed and Vector Blue AP colour substrate was added to each tissue section for 10 min at room temperature. Vector Blue AP colour substrate was washed off in TBS under gentle agitation for 5 min. For HRP dependent detection, the NovaRED HRP substrate kit (SK-4800, Vector Laboratories, Burlingame, CA, USA) was used. This was prepared in accordance with the instructions of the manufacturer. Following washing, excess TBS was removed and NovaRED HRP substrate was added to each tissue section for 20 min at room temperature. The substrate was washed off in TBS under gentle agitation for 5 min.
In experiments where both primary antibodies were raised in the same species, a sequential staining protocol was used, where an antibody denaturation step 'antigen stripping' was used before the application of the 2nd primary antibody [19]. Thus, following Vector Blue AP substrate development, sections underwent a second heat-induced epitope retrieval (HIER) for antibody denaturation: Tris-EDTA buffer (10 mM Trizma base (Sigma Aldrich, Poole, UK) 1 mM EDTA solution (Sigma Aldrich, Poole, UK), 0.05% Tween 20 (Sigma Aldrich, Poole, UK), pH 9.0) for 5 min at 100 • C, by being microwaved in a pressure cooker. Once the pressure dropped, the lid of the pressure cooker was removed, and slides were cooled under cold running water for 5 min. It was noted that the first chromogen needed to be Vector Blue because the NovaRED substrate did not withstand the denaturation process.
A schematic diagram shows the distinctions between the monoplex and multiplex procedures ( Figure 1). moved, and slides were cooled under cold running water for 5 min. It was noted that the first chromogen needed to be Vector Blue because the NovaRED substrate did not withstand the denaturation process.
A schematic diagram shows the distinctions between the monoplex and multiplex procedures ( Figure 1).

Figure 1. Schematic of single-stain and multiplex immunohistochemistry reporter systems.
In single-stained immunohistochemistry (IHC) (above), neutrophils and platelets are detected in separate tissue sections using a horse radish peroxidase (HRP)-dependent reporting system. The 3,3'diaminobenzidine (DAB) substrate forms a brown precipitate in positively stained regions. In multiplex IHC (below), platelets and neutrophils can be detected in the same tissue section. Platelets are detected using a HRP-dependent reaction, whilst neutrophils are detected using an alkaline phosphatase (AP) dependent reaction. Blue and red colour substrates allow for visualisation of colocalised events.

Counterstaining, Dehydration and Clearing
Sections were submerged into Hematoxylin Solution, Gill No. 1, for 2 min before being washed in tap water (monoplex only). Sections were briefly submerged in differentiation solution for 3 s, before being washed for a final time in tap water. Sections were dehydrated in increasing concentrations of ethanol (70%, 90% and 100%) by being submerged for 1 min in each ethanol concentration (monoplex), or 10 s in each ethanol concentration, to avoid solubilising colour substrates (multiplex). Sections were cleared in Figure 1. Schematic of single-stain and multiplex immunohistochemistry reporter systems. In single-stained immunohistochemistry (IHC) (above), neutrophils and platelets are detected in separate tissue sections using a horse radish peroxidase (HRP)-dependent reporting system. The 3,3'-diaminobenzidine (DAB) substrate forms a brown precipitate in positively stained regions. In multiplex IHC (below), platelets and neutrophils can be detected in the same tissue section. Platelets are detected using a HRP-dependent reaction, whilst neutrophils are detected using an alkaline phosphatase (AP) dependent reaction. Blue and red colour substrates allow for visualisation of colocalised events.

Counterstaining, Dehydration and Clearing
Sections were submerged into Hematoxylin Solution, Gill No. 1, for 2 min before being washed in tap water (monoplex only). Sections were briefly submerged in differentiation solution for 3 s, before being washed for a final time in tap water. Sections were dehydrated in increasing concentrations of ethanol (70%, 90% and 100%) by being submerged for 1 min in each ethanol concentration (monoplex), or 10 s in each ethanol concentration, to avoid solubilising colour substrates (multiplex). Sections were cleared in 100% xylene for 10 min and cover-slipped with DPX mounting medium (monoplex) or cleared with Histoclear for 10 min and cover-slipped with Vectamount, a non-xylene based mounting medium (multiplex) used in histology. Images were captured using an LED upright bright-field microscope (DM2000 Leica, Nussloch, Germany) attached to a bright-field microscope camera (DCF295, Nussloch, Germany).

Protocol Optimisation for IHC in Rat Tissues to Identify Neutrophils and Platelets Using Singleplex Protocols
Protocol optimisation experiments were performed to identify a suitable methodology for IHC to detect platelets and neutrophils in rat tissues. Due to the limited number of reagents available that are specific for the rat, cross-reactivity with antibodies that have been made to target human and mouse antigens were tested. Both formalin-fixed paraffin-wax-embedded (FFPE) and frozen tissues were also tested, with different antigen retrieval buffers tested for the FFPE tissues. Antibodies were tested on FFPE samples initially, since this is the preferred method for optimal preservation of tissue morphology. When antibodies failed in FFPE tissue, for example, all antibodies tested for rat platelets, we tested antibodies in frozen tissues. For the PNC staining, it was essential that both antibodies were suitable for the same sample-preparation type. Several concentrations of each antibody were tested, determined through the published literature and user reviews on the manufacturers' websites.
Several primary antibodies were tested to detect neutrophils in rat tissues ( Table 1). The only successful neutrophil-specific antibody out of those tested was an anti-myeloperoxidase (MPO) antibody (ab9535, Abcam, Cambridge, UK). This anti-MPO antibody was detected using an anti-rabbit biotinylated secondary antibody (BA1000, Vector Laboratories, Burlingame, CA, USA), Vectastain Elite ABC HRP detection kit (PK-6100, Vector Laboratories, Burlingame, CA, USA) and DAB development buffer (Sigma Aldrich, Poole, UK). The antibody worked effectively in both paraffin-wax-embedded and frozen-tissue sections ( Figure 2A). No unspecific binding of detection reagents was observed in a consecutive section that received no primary antibody, indicating no unspecific binding of detection reagents ( Figure 2B). A haematoxylin nuclear counterstain revealed that positively stained regions were associated with cells featuring a multi-lobed nucleus, a key characteristic of neutrophils ( Figure 2C). Neutrophils could be detected proximal to blood vessels in the lung ( Figure 2D). As MPO is an enzyme secreted from neutrophils upon degranulation, extracellular MPO positivity was also observed, particularly in the liver and within focal necrotic regions ( Figure 2E,F).
Primary antibodies were also tested to detect platelets in rat tissues (Table 2). Antibodies were selected based on those already successfully used by the Sackler Institute of Pulmonary Pharmacology at King's College London, in the published literature and per user reviews on supplier websites. The only successful platelet-specific antibody out of those tested was an anti-CD61 antibody (554951, BD Biosciences, Franklin Lakes, NJ, USA), but this only worked when used with frozen tissue. This anti-CD61 antibody was detected using an anti-rabbit biotinylated secondary antibody (BA1000, Vector Laboratories, Burlingame, CA, USA), Vectastain Elite ABC horseradish peroxidase (HRP) detection kit (PK-6100, Vector Laboratories, Burlingame, CA, USA) and DAB development buffer. Platelets could be observed within blood vessels and within the lung parenchyma ( Figure 3A). No unspecific binding of detection reagents was observed in a consecutive section that received all detection reagents with no primary antibody ( Figure 3B). Platelets could be observed within both lung tissues ( Figure 3C,D) and liver tissues ( Figure 3E,F).
Protocol optimisation for immunohistochemistry in rat tissues identified PNCs using a multiplex protocol. As the only suitable platelet specific antibody worked solely in frozen-tissue sections, the detection of PNCs was limited to analysis of tissue sections in this format. To detect PNCs, both primary platelet and neutrophil antibodies (anti-MPO and anti-CD61) were incubated together on the same section. As each antibody was raised in a different species, different detection systems could be used simultaneously to detect the primary antibodies. Primary antibodies were, therefore, detected using a HRP-dependant reaction and an AP-dependant reaction. As PNCs are associated with colocalisation of both cell types, blue and red detection systems were chosen to detect any overlapping events that may not be visible using DAB substrate (Table 6). PNC staining was observed in both the rat lung ( Figure 4A,B) and liver tissues ( Figure 4C,D). Primary antibodies were also tested to detect platelets in rat tissues (Table 2). Antibodies were selected based on those already successfully used by the Sackler Institute of Pulmonary Pharmacology at King's College London, in the published literature and per user reviews on supplier websites. The only successful platelet-specific antibody out of those tested was an anti-CD61 antibody (554951, BD Biosciences, Franklin Lakes, NJ, USA), but this only worked when used with frozen tissue. This anti-CD61 antibody was detected using an anti-rabbit biotinylated secondary antibody (BA1000, Vector Laboratories, Burlingame, CA, USA), Vectastain Elite ABC horseradish peroxidase (HRP) detection kit (PK-6100, Vector Laboratories, Burlingame, CA, USA) and DAB development buffer. Platelets could be observed within blood vessels and within the lung parenchyma ( Figure  3A). No unspecific binding of detection reagents was observed in a consecutive section that received all detection reagents with no primary antibody ( Figure 3B). Platelets could be observed within both lung tissues ( Figure 3C,D) and liver tissues ( Figure 3E,F).   Protocol optimisation for immunohistochemistry in rat tissues identified PNCs a multiplex protocol. As the only suitable platelet specific antibody worked solely i zen-tissue sections, the detection of PNCs was limited to analysis of tissue sections i format. To detect PNCs, both primary platelet and neutrophil antibodies (anti-MPO anti-CD61) were incubated together on the same section. As each antibody was rais a different species, different detection systems could be used simultaneously to dete primary antibodies. Primary antibodies were, therefore, detected using a HRP-depe reaction and an AP-dependant reaction. As PNCs are associated with colocalisati both cell types, blue and red detection systems were chosen to detect any overlap events that may not be visible using DAB substrate (Table 6). PNC staining was obs in both the rat lung ( Figure 4A,B) and liver tissues ( Figure 4C,D).

Protocol Optimisation for IHC to Identify Neutrophils and Platelets in Pig Tissues
Protocol optimisation experiments were performed to identify a suitable methodology for IHC to detect platelets and neutrophils in pig tissues. Several antibodies were tested, based on the literature reviews and user reviews on supplier websites ( Table 3). The only successful neutrophil specific antibody out of those tested was an anti-MPO antibody (ab9535, Abcam, Cambridge, UK), the same antibody we had identified as successful for IHC in rat tissues, as described above. The antibody worked effectively in both FFPE and frozen issue; as pig platelets could also be detected in FFPE, and this method optimally preserves tissue morphology, we focused on the FFPE neutrophil staining ( Figure 5A). No unspecific binding of detection reagents was observed in a consecutive section that received no primary antibody ( Figure 5B). Like for the rat tissue, neutrophils could be visualised within blood vessels in the lung (Figure 5C,D) and were localised to focal necrotic regions in the liver ( Figure 5E,F).   Several primary antibodies were tested to detect platelets in pig tissues (Table 4). Antibodies were selected based on the published literature and user reviews on supplier websites. Out of those tested, two antibodies showed positive platelet staining, which were an anti-CD42b antibody (554951, BD Biosciences, Franklin Lakes, NJ, USA) and an anti-CD49b antibody (ab181548, Abcam, Cambridge, UK). The anti-CD42b antibody worked in both FFPE tissue and frozen tissue, whilst the anti-CD49b antibody only worked in frozen tissue (Figures 6 and 7). Platelets were observed within pulmonary blood vessels ( Figures  6A and 7A). No unspecific binding of detection reagents was observed in consecutive sections ( Figures 6B and 7B). Platelets were identified with both antibodies in pulmonary blood vessels ( Figure 6C), which were not present in the negative reagent control (Figure 6D), as well as multi-lobular nucleated cells ( Figure 7C), plus there were platelet aggregates in regions of alveolar haemorrhage ( Figure 7D). Platelets were detected in the liver with both antibodies ( Figure 6E,F and Figure 7E,F).

Protocol Optimisation for IHC in Porcine Tissues to Identify PNCs Using a Multiplex Protocol
Platelet and neutrophil antibodies were effective in both FFPE and frozen tissue. However, since FFPE tissue preparation retains better tissue morphology than frozen tissue, PNCs were detected in FFPE tissue. However, since both platelet (anti-CD42b) and neutrophil (anti-MPO) antibodies were both raised in rabbits, a method was developed based on the previous literature [20]. Instead of adding primary antibodies together, the neutrophil (anti-MPO) primary antibody, AP detection reagents and AP colour substrate (blue) were added, before an additional HIER step to denature the residual antibody and reagents. This step was followed by addition of the platelet (anti-CD42b) antibody, HRP detection reagents and HRP colour substrate (red). It was critical to use the colour substrates in this order, because the red substrate is not heat stable. PNCs are associated with colocalisation of both cell types, so blue and red detection systems were chosen to detect any overlapping events that may not be visible with DAB substrate. Lung CD42b can be observed in red (Figure 8A), and lung MPO can be observed in blue ( Figure 8B). When multiplexed, colocalised events could be observed in the lung (Figure 8C,D) and liver ( Figure 8E,F).

Protocol Optimisation for IHC to Identify Neutrophils and Platelets in Pig Tissues
Protocol optimisation experiments were performed to identify a suitable methodology for IHC to detect platelets and neutrophils in pig tissues. Several antibodies were tested, based on the literature reviews and user reviews on supplier websites ( Table 3). The only successful neutrophil specific antibody out of those tested was an anti-MPO antibody (ab9535, Abcam, Cambridge, UK), the same antibody we had identified as successful for IHC in rat tissues, as described above. The antibody worked effectively in both FFPE and frozen issue; as pig platelets could also be detected in FFPE, and this method optimally preserves tissue morphology, we focused on the FFPE neutrophil staining (Figure 5A). No unspecific binding of detection reagents was observed in a consecutive section that received no primary antibody ( Figure 5B). Like for the rat tissue, neutrophils could be visualised within blood vessels in the lung (Figure 5C,D) and were localised to focal necrotic regions in the liver ( Figure 5E,F). Several primary antibodies were tested to detect platelets in pig tissues (Table 4). Antibodies were selected based on the published literature and user reviews on supplier websites. Out of those tested, two antibodies showed positive platelet staining, which were an anti-CD42b antibody (554951, BD Biosciences, Franklin Lakes, NJ, USA) and an anti-CD49b antibody (ab181548, Abcam, Cambridge, UK). The anti-CD42b antibody worked in both FFPE tissue and frozen tissue, whilst the anti-CD49b antibody only worked in frozen tissue (Figures 6 and 7). Platelets were observed within pulmonary blood vessels (Figures 6A and 7A). No unspecific binding of detection reagents was observed in consecutive sections (Figures 6B and 7B). Platelets were identified with both antibodies in pulmonary blood vessels ( Figure 6C), which were not present in the negative reagent control (Figure 6D), as well as multi-lobular nucleated cells ( Figure 7C), plus there were platelet aggregates in regions of alveolar haemorrhage ( Figure 7D). Platelets were detected in the liver with both antibodies (Figures 6E,F and 7E,F).

Protocol Optimisation for IHC in Porcine Tissues to Identify PNCs Using a Multiplex Protocol
Platelet and neutrophil antibodies were effective in both FFPE and frozen tissue. However, since FFPE tissue preparation retains better tissue morphology than frozen tissue, PNCs were detected in FFPE tissue. However, since both platelet (anti-CD42b) and neutrophil (anti-MPO) antibodies were both raised in rabbits, a method was developed based on the previous literature [20]. Instead of adding primary antibodies together, the neutrophil (anti-MPO) primary antibody, AP detection reagents and AP colour substrate (blue) were added, before an additional HIER step to denature the residual antibody and reagents. This step was followed by addition of the platelet (anti-CD42b) antibody, HRP detection reagents and HRP colour substrate (red). It was critical to use the colour substrates in this order, because the red substrate is not heat stable. PNCs are associated with colocalisation of both cell types, so blue and red detection systems were chosen to detect any overlapping events that may not be visible with DAB substrate. Lung CD42b can be observed in red ( Figure 8A), and lung MPO can be observed in blue ( Figure 8B). When multiplexed, colocalised events could be observed in the lung ( Figure 8C,D) and liver ( Figure 8E,F).

Discussion
Advances in trauma care, particularly with regard to haemorrhage control a suscitation, have resulted in increased survival for the severely injured. However survivors develop multi-organ dysfunction syndrome (MODS) and failure due to t temic response to injury and the development of SIRS (systemic inflammatory re syndrome). There is, therefore, an interest in better understanding MODS after trauma [21][22][23]. It is important to understand the origins of MODS, including the i matory mechanisms contributing to MODS, which in part requires the assessm

Discussion
Advances in trauma care, particularly with regard to haemorrhage control and resuscitation, have resulted in increased survival for the severely injured. However, many survivors develop multi-organ dysfunction syndrome (MODS) and failure due to the systemic response to injury and the development of SIRS (systemic inflammatory response syndrome). There is, therefore, an interest in better understanding MODS after severe trauma [21][22][23]. It is important to understand the origins of MODS, including the inflammatory mechanisms contributing to MODS, which in part requires the assessment of tissues from animals following traumatic injury, including blast injury. However, the availability of reagents to investigate inflammatory pathways in the animal species used for modelling blast injury, namely the rat and pig, is poor, with most reagents being developed for use in mice or humans. In the present study, we have, therefore, carried out an extensive search and evaluation of commercially available reagents to detect platelets and neutrophils in tissues using IHC, to determine whether any of these could be suitable to evaluate inflammatory changes in rats and pigs that have been exposed to blast injury, which could be used in the future in these species to better understand the inflammatory chances associated with MODS.
To determine inflammatory cell recruitment into lung and liver in rat and pig models of traumatic haemorrhagic shock, with and without thoracic blast injury, we have evaluated various anti-human and anti-mouse antibodies for cross-reactivity, which have been reported to target platelets and neutrophils in our laboratory and others [2,[24][25][26][27]. Furthermore, we have investigated the use of these antibodies to detect PNCs as the published literature describing histological protocols for detecting platelets and neutrophils in porcine and rodent tissues is limited.
An anti-human MPO antibody yielded successful and specific neutrophil staining of tissues from both species, using frozen and FFPE tissues. Although MPO is a neutrophil activation marker, positive staining was mostly observed intracellularly, suggesting it was of value in enumerating neutrophils in tissues. For the detection of platelets in pig tissues, an antibody recognising CD49b worked well in frozen tissue, whilst CD42b worked in both frozen and FFPE tissues. This anti-CD42b antibody yielded similar staining results in the FFPE pig tissue to those observed in FFPE murine lung tissue in a recent study by our laboratory [2]. We have previously used a CD42b antibody for platelet quantification because 1) FFPE-fixed tissues retain better morphology, and 2) FFPE tissue fixation and processing techniques also allow for retention of the RBCs in the tissues [28]. This is of interest, given that alveolar haemorrhage is a major feature of blast injury, and we have previously reported that red blood cells (and their products) can induce an inflammatory response in the lung, which is dependent on platelets [28]. However, surprisingly, antimouse antibodies recognising CD42b and CD49b did not adequately stain platelets in rat tissues, so we have still to identify a suitable platelet antibody for use in FFPE rat tissue. However, during the protocol optimisation period, another laboratory reported rat platelet staining using an anti-CD61 antibody for immunofluorescence microscopy [29]. Whilst this antibody was also successful in staining for platelets in frozen rat tissue in a blast-injury model, it was not suitable for use in FFPE tissue, despite testing several different antigen retrieval conditions. Importantly, given that platelets and neutrophils are known to co-operate during inflammatory processes and often form PNCs, we could also quantify the presence of PNCs in tissues using the same antibodies that were used for single-stain IHC. We were successful in the development of a multiplex IHC protocol that enabled dual colour detection of double-positive, colocalised events.
Whilst chromogenic IHC facilitates investigation into cellular recruitment and resolves spatial information of cells and their colocalisation, there are some limitations of the technique. Whilst cells can be enumerated manually or be measured digitally using image analysis (IA) software, chromogenic IHC is a semiquantitative method due to the signal amplification steps involved. An amplification of signal by IHC is also a limitation of quantifying cellular colocalisation, as this may cause proximity of proteins to be exaggerated, thus creating false positive events. An alternative method that may be useful to observe colocalised events such as PNCs is a proximity ligation assay (PLA), as, using this methodology, a signal is only observed when two proteins of interest are expressed at the same cellular location [30]. In addition, multiplex chromogenic IHC is also limited by overlapping colours in the brightfield light spectra, which can be overcome by using multiplex immunofluorescence (mIF). Advances in mIF have enabled analysis of up to eight biomarkers in just one tissue section, which is achievable using both automated and manual staining methods [31]. Furthermore, emerging technologies are making it possible to measure up to 100 markers in one tissue section, with the capability to overlay both RNA and protein expression [32].
In conclusion, the methods described here will help characterise the nature of plateletneutrophil interactions in species used to model the pathology of blast-trauma-related injury, a heterotypical interaction that may have multiple consequences and temporal distinctions across different body compartments [2,33,34], as well as being of value for the investigation of platelets and neutrophils in other experimental models in rats and pigs.
Author Contributions: S.A. designed and performed the research, analysed the data and wrote the paper. S.W. and E.K. provided tissue. S.W., E.K., S.C.P. and C.P.P. proposed the project, designed the research and wrote the paper. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Defence Science and Technology Laboratory (DSTL), UK, with funds awarded to C.P.P. and S.C.P., and by an endowment from the Sackler Foundation, awarded to C.P.P.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.